Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in humans and other mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a contributing factor to regeneration failure in mammals. We demonstrate that the SLRPs chondroadherin, fibromodulin, lumican, and prolargin are enriched in rodent and human but not zebrafish CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer mechano-structural properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as inhibitory ECM factors that impair axon regeneration by modifying tissue mechanics and structure, and identifies their enrichment as a feature of human brain and spinal cord lesions. These findings imply that SLRPs may be targets for therapeutic strategies to promote CNS regeneration.


Supplementary Fig. 1 | Mass spectrometry-based quantitative proteomics reveals changes in
ECM composition during zebrafish spinal cord regeneration.
a-b) Principle component analysis of mass spectrometry-based proteomics data using quantitative values of all identified proteins across samples.Lesioned samples (1 dpl, 2 dpl) cluster distinct from unlesioned age-matched control samples (4 dpf, 5 dpf).Each data point represents one independent biological replicate.
c-f) Volcano plots of all quantified proteins for the given analyses (1 dpl vs. unlesioned age-matched controls, (c, e); 2 dpl vs. unlesioned age-matched controls, (d, f)) with their log 2 -transformed ratios of the mean-centered abundances (FC, fold change) and -log 10 -transformed P-values (two-tailed t-test).Dashed lines indicate the threshold of a permutation-based FDR correction for multiple hypotheses (FDR < 0.1, s 0 = 0.1) for identification of significantly altered abundances.Proteins with significantly altered abundance were further filtered by |FC| ≥ 1.3.Area highlighted (blue) in (c) and ( d) is shown at a different scale in (e) and (f).
g-h) Reactome pathway analysis of differentially enriched proteins reveals ECM-associated terms (blue) being overrepresented at 1 dpl (g) and 2 dpl (h).Bonferroni correction.b) Fold change expression of indicated genes in the zebrafish spinal lesion site at 1 dpl over unlesioned age-matched controls, as determined by qRT-PCR.Expression of indicated genes coding for neurocan b (Ncanb) and basal lamina constituents is not upregulated in the zebrafish spinal lesion site at 1 dpl.Note that cthrc1a served as a positive control for the detection of injuryinduced expression of genes coding for ECM proteins    b) mCherry fluorescence (black) is robustly detected following induction of indicated slrp-mCherry fusions in pdgfrb + cells in pdgfrb:TetA;TetRE:SLRP-mCherry (short pdgfrb:SLRP) transgenic zebrafish.Note that the SLRP-mCherry fusions are secreted proteins and that the pattern of mCherry fluorescence resembles that of the pdgfrb + cell niche in pdgfrb:GFP transgenic animals, labeling mainly the myosepta and vasculature (compare (a) and (c)).Also note that the pattern of protein deposition varies among different SLRPs and that some exhibit mesh and fiber-like structures (arrowheads in magnified views; lookup table is shown).Similar results were obtained in n≥10 animals for each experimental condition.
c) pdgfrb + cells (black) in pdgfrb:GFP transgenic animals.Note that GFP is located in the cytoplasm.Similar results were obtained in n≥10 animals for each experimental condition.
d) pdgfrb + cell-specific induction of indicated slrp-mCherry fusions leads to pericellular labeling (magenta) of pdgfrb + cells (green) in pdgfrb:SLRP;pdgfrb:GFP transgenic animals.Note that the SLRP-mCherry fusions are secreted proteins and that the GFP protein is located in the cytoplasm.Similar results were obtained in n≥10 animals for each experimental condition.
g) pdgfrb + cell-specific induction of the SLRPs chad, fmoda, lum, or prelp but not aspn in pdgfrb:SLRP transgenic zebrafish reduces the thickness of the axonal bridge (white; analyzed in elavl3:GFP-F transgenics).Shown are example images of axonal bridges quantified in Fig. 5g.permutation-based FDR correction for multiple hypotheses (FDR < 0.1, s 0 = 0.1) for identification of significantly altered abundances.Proteins with significantly altered abundance were further filtered by |FC| ≥ 1.3.Since the MS analysis cannot discriminate between endogenous and ectopic SLRP proteins, the respective manipulated protein has been excluded from the volcano plot.Indicated proteins were selected for verification by in situ hybridization (ISH; see (d)).
d) Induction of the SLRPs chad, fmoda, lum, or prelp in pdgfrb:SLRP transgenic zebrafish does not lead to major changes in the expression (blue) of indicated genes in the lesion site at 1 dpl, as determined by ISH.Transcript levels of five genes coding for matrisome proteins that showed the highest (yet non-significant) fold change in each of the four experimental conditions in (c) were evaluated.
where  is the refractive index increment in mL/g, i.e. the slope of a d d diagram.Finally, we arrive at an expression of the mass density in dependence of the refractive index as This expression is not exact since  1/ is not generally given.

i)
Expression of the indicated genes in the trunk of 4 dpf larvae, as determined by in situ hybridization.Images shown are age-matched unlesioned controls for the lesioned animals shown in Fig. 1e (lateral view; rostral is left).n≥6 for each gene.Scale bars: 100 µm.a-i) dpl, days post-lesion; expl.var., explained variance; FC, fold change; FDR, false discovery rate; padj, adjusted p value.The Source data are provided as a Source Data file.Supplementary Fig. 2 | Neurocan, basal lamina components, and small leucine-rich proteoglycans are not enriched in the zebrafish spinal lesion site.a) Comparative proteomics analysis reveals differentially enriched matrisome proteins between rat (black) and zebrafish (red) after SCI.Shown are proteins that exhibit a high abundance (FDR < 0.1, FC  1.3) after SCI in zebrafish but a low abundance (FDR < 0.1, FC  -1.3 | n.s.) in the rat spinal lesion site.Each data point represents one biological replicate.Data are means ± SEM.

f)
Mean fold change values of indicated genes and proteins at 1 dpl over unlesioned age-matched controls as determined by qRT-PCR (Fig.2b) and mass spectrometry-based quantitative proteomics (Supplementary Fig.1c) are highly correlated (R² = 0.9443).Pearson correlation.Fold change values are presented in log scale.g) Transcripts of indicated genes (blue) are detectable by in situ hybridization in whole-mount zebrafish larvae at 4 dpf.Images shown are lateral views of the heads of animals depicted in Fig. 2c.a-g) Scale bars: 100 µm (g), 50 µm (c).dpf, days post-fertilization; dpl, days post-lesion; FC, fold change; FDR, false discovery rate; n.s., not significant; wpl, weeks post-lesion.The rat icon in panels (a) and (d) was created using BioRender.Source data are provided as a Source Data file.Supplementary Fig. 3 | SLRPs are enriched in human brain lesions.a-c) Immunohistochemical examination of human brain specimens.Hematoxylin and eosin (H&E) staining, and 3,3′-diaminobenzidine (DAB) staining of anti-GFAP, anti-NeuN, and anti-LUM antibodies on scarred brain tissue from patients with previous surgery (re-OP, a) or traumatic brain injury (TBI, b), and no scar control brain tissue (c).Areas of scarring were identified by H&E staining pattern and absence of immunoreactivity of the neuronal marker anti-NeuN.Anti-LUM immunoreactivity is increased in areas of scarring caused by previous surgery (a), contusion (arrowheads in b), or local hemorrhage (asterisk in b).Anti-LUM immunoreactivity is negligible in healthy human brain autopsy tissue with no signs of scarring (c).Shown are coronal sections.Size of scale bars is given in the figure.Six cases with scars following TBI or previous surgery, and six cases without scars were analyzed and showed similar results (Supplementary a-d)The human icon in panels (a), (b), (c), and (d) was created using BioRender.Supplementary Fig.4| pdgfrb + cell-specific targeting of SLRPs in zebrafish.a) pdgfrb + myoseptal (arrows) and perivascular (arrowhead) cells (green) are a major source of endogenous SLRPs in uninjured larval zebrafish.Shown is the fold change expression of indicated genes in FACS-isolated GFP + cells over GFP − cells in trunk tissue of pdgfrb:GFP transgenic animals at 4 dpf, as determined by qRT-PCR.Note that pdgfrb served as positive control for the enrichment of pdgfrb + cells.Fold change values are presented in log scale.Each data point represents one independent biological replicate.

a- h )
Images shown are maximum intensity projections or 3D reconstructions (d) of unlesioned trunk or lesion site (lateral view; rostral is left).Data are means ± SEM.Scale bars: 100 µm (a, e, f), 50 µm (b, c), 25 µm (b (magnified view), g, h), 10 µm (d).d, days; DOX, doxycycline.Source data are provided as a Source Data file.Supplementary Fig. 5 | Neuron-specific targeting of SLRPs in zebrafish.a) The Xla.Tubb promoter drives transgene expression in all neurons of the brain and spinal cord 2 .Image shown is a maximum intensity projection of a Xla.Tubb:DsRed transgenic zebrafish larva at 3 dpf (rostral is left; dorsal is up).Fluorescence signal of the cytoplasmic DsRed protein is indicated in black.b) Spinal cord transection leads to an acute neuronal loss, as indicated by the lack of DsRed fluorescence (black) in the lesion center of Xla.Tubb:DsRed transgenic animals at 8 hpl.Note that low fluorescence signal can be detected in the non-neuronal lesion core at 24 hpf due to the presence of cytoplasmic DsRed protein in regenerating axonal fibers (arrowhead).Images shown are maximum intensity projections of unlesioned trunk or lesion site (lateral view; rostral is left).Similar results were obtained in n≥10 animals.c) mCherry fluorescence (black) is robustly detected and largely confined to the brain and spinal cord following induction of indicated slrp-mCherry fusions in neurons in XlaTubb:TetA;TetRE:SLRP-mCherry (short Xla.Tubb:SLRP) transgenic zebrafish.Note that the lesion core is largely devoid of fluorescence signal due to the absence of neuronal somata at 24 hpl (compare (b)).Also note the granular fluorescence pattern due to the extracellular localization of the SLRP proteins.Images shown are maximum intensity projections of the head, unlesioned trunk, or lesion site (lateral view; rostral is left).Similar results were obtained in n≥10 animals for each experimental condition.d) Xla.Tubb promoter-driven expression of either DsRed (Xla.Tubb:DsRed) or indicated slrp-mCherry fusions (Xla.Tubb:SLRP) leads to different fluorescent labeling patterns (magenta) in the larval zebrafish spinal cord.Fluorescence signal is detected in the central canal and its ependymal lining in Xla.Tubb:SLRP (arrowheads) but not in Xla.Tubb:DsRed transgenic animals.Note that the DsRed protein is localized to the cytoplasm whereas SLRPs are secreted proteins.Also note that the Xla.Tubb promotor does not drive expression in ependymo-radial glia cells surrounding the central canal.Shown are magnified views of the images presented in Fig. 6c (transversal views of the spinal cord; dorsal is up).Similar results were obtained in n≥10 animals for each experimental condition.e) Neuron-specific induction of indicated slrp-mCherry fusions leads to pericellular labeling (arrowheads; magenta) of primary neurons (green) prepared from dissociated Xla.Tubb:SLRP;elavl3:GFP-F transgenic animals.Note that the GFP protein is membranelocalized.Images shown are single optical sections at the level of the substrate-cell-interface.Similar results were obtained in n≥10 neurons for each experimental condition.f) Neuron-specific induction of the SLRPs chad, fmoda, lum, or prelp in Xla.Tubb:SLRP transgenic zebrafish does not reduce the thickness of the axonal bridge (white; analyzed in elavl3:GFP-F transgenics) at 2 dpl.Shown are example images of axonal bridges quantified in Fig. 6d.Images shown are maximum intensity projections of the lesion site (lateral view; rostral is left).a-f) Scale bars: 500 µm (a), 100 µm (b, c), 25 µm (f), 10 µm (d), 5 µm (e), 1 µm (magnified view in e).d; days; dpf, days post-fertilization; DOX, doxycycline; hpl, hours post-lesion.

Table 1
Anti-LUM, anti-PRELP, anti-CHAD, and anti-FMOD immunoreactivity is negligible in healthy human brain autopsy tissue with no signs of scarring.Images shown are immunofluorescence controls for data shown in Fig.3.Six cases without scars were analyzed and showed similar results (Supplementary Table1).Shown are coronal sections.Scale bars: 500 µm.

Table 3 | Summary of results from immunofluorescence stainings on human spinal cord samples. Case ID Increased anti-CHAD immunoreactivity in E segment 1 as compared to R/C 2 segment? Increased anti-FMOD immunoreactivity in E segment 1 as compared to R/C 2 segment? Increased anti-LUM immunoreactivity in E segment 1 as compared to R/C 2 segment? Increased anti-PRELP immunoreactivity in E segment 1 as compared to R/C 2 segment?
The number of animals displaying the phenotype and the total number of animals is given.Black arrowheads indicate ISH signal in the center of the lesion site, white arrowheads indicate absence of ISH signal.Images shown are brightfield recordings of the lesion site (lateral view; rostral is left).Scale bars: 100 µm.
a-d) d, days; DOX, doxycycline; expl.var., explained variance; FC, fold change; FDR, false discovery rate.Source data are provided as a Source Data file.